Correlation of PDCD5 and Apoptosis in Hair Cells and Spiral Ganglion Neurons of Different Age of C57BL/6J Mice

This study examined the expression pattern of programmed cell death 5 (PDCD5) in co-chlear hair cells and spiral ganglion neurons (SGNs) and its association with age-related hearing loss in mice.Sixty C57BL/6J (C57) mice at different ages were divided into four groups (3,6,9 or 12 months).PDCD5 expression was detected by using immunohistochemistry,real-time PCR and Western blot.Morphological change of the cochleae was also evaluated by using immunoassay.The results showed that the expression of PDCD5 had a gradual increase with ageing in both protein and RNA levels in C57 mice,as well as gradually increased apoptosis of cochlear hair cells and SGNs.In addition,we also found that caspase-3 activity was enhanced and its expression was enhanced with ageing.It is implied that overexpression of PDCD5 causes the increase in caspase-3 activity and the subsequent increase of apoptosis in cochlear hair cells and SGNs,and thereby plays a role in the pathogenesis of presbycusis.Thus,PDCD5 may be a new target site for the treatment and prevention of age-related hearing loss.

Gamma-aminobutyric acid A receptor and N-methyl-D-aspartate receptor subunit expression in rat spiral ganglion neurons

BACKGROUND： Gamma-aminobutyric acid A （GABAA） and N-methyl-D-aspartate （NMDA） receptors are significant receptors in the central nervous system. An understanding of GABAA and NMDA receptor expression in spiral ganglion neurons （SGN） provides information for the functional role of these receptors in the auditory system. OBJECTIVE： To investigate mRNA expression of GABAA receptor （GABAAR） and NMDA receptor （NMDAR） subunits in the rat SGN. DESIGN, TIME AND SETTING： This in vitro, molecular biological study was performed at the Laboratory of Otolaryngology-Head and Neck Surgery, Guangxi Medical University, China from July 2007 to May 2008. MATERIALS： Reverse Transcriptase Kit and Taq DNA polymerase were purchased from Fermentas Burlington, ON, Canada; GABAAR and NMDAR primers were purchased from Shanghai Sangon, Shanghai, China. METHODS： SGN from 3-5 day postnatal Wistar rats was collected for primary cultures, mRNA expression of GABAAR and NMDAR subunits in the SGN was determined by reverse transcription polymerase chain reaction. MAIN OUTCOME MEASURES： Expression levels of GABAAR and NMDAR subunits were determined by quantitative analysis. RESULTS： GABAAR subunits （αl 6, β1 3, and y1 3） and NMDAR subunits （NR1, NR2A, NR2B, NR2C, NR2D, NR3A, and NR3B） were detected in the SGN. In α subunit genes of GABAAR, α1 and α3 expression was similar （P 〉 0.05） and greater than the other subunits. Of the β subunit genes, β1 subunit mRNA levels were greater than β2 and β3. Of the y subunit genes, y2 subunit mRNA levels were greater than y1 and y3. NR1 mRNA expression was the greatest of NMDAR subunits. CONCLUSION： GABAAR subunits （α1 6, β1-3, and y1-3） and NMDAR subunits （NR1, NR2A, NR2B, NR2C, NR2D, NR3A, and NR3B） were expressed in the rat SGN. Through comparison of GABAAR and NMDAR subunit expression, possible GABAAR combinations, as well as highly expressed subunit combinations, were estimated, which provided information for pharmacological and electrophysiological characteristics of GABAAR in the auditory system.

Salicylate enhances expression and function of NMDA receptors in cochlear spiral ganglion neurons

Objective To study the effect of salicylate on the expression and function of NMDA receptors in spiral ganglion neurons （SGNs）. Methods The mRNA of NR1 subunit of NMDA receptor in modiolus tissues were detected by Real time fluorescence quantitative PCR （FQ-PCR）. NMDA receptor whole-cell currents were recorded using patch clamp in acute isolated SGNs. Results Compared with the control group, salicylate significantly increased the mRNA level of NR1 subunit in SGNs. NMDA of concentrations ranging from 0.1 mM to 10 mM evoked no current in SGNs. NMDA （0. 1mM and 0.5 mM） applied with salicylate （5 mM）, however, induced inward currents （212.6±15.2pA, n=5; 607.9±44.3pA, n=5） in a dose-dependent manner, which could be inhibited by APV. Salicylate alone did not produce any current in SGNs. Conclusion Salicylate increases the expression of NMDA receptors and facilitates the currents mediated by NMDA receptors in SGNs.

(-)-Epigallocatechin-3-gallate protects spiral ganglion neurons against amikacin-induced apoptosis

Morphology of spiral ganglion neurons （SGNs） in Sprague-Dawley rats before and after amikacin treatment was observed by transmission electron microscopy. Amikacin induced cochlear SGN apoptosis. Immunohistochemical staining and RT-PCR revealed a decrease in Bcl-2 protein ex-pression, and an increase in Bax protein, caspase-3 protein and caspase-6 mRNA expression fol-lowing amikacin treatment. （-）-Epigallocatechin-（3）-gallate （EGCG） inhibited SGN Bax protein, caspase-3 protein and caspase-6 mRNA expression, and enhanced Bcl-2 protein expression, thereby decreasing SGN apoptosis. Results demonstrated that EGCG can protect SGNs against amikacin-induced injury.

Phenotypic differentiation of neonatal rat cochlear spiral ganglion neurons following trypsin dissociation and culture

BACKGROUND： Under laboratory conditions, cochlear spiral ganglion neurons are commonly isolated and cultured by mechanical dissociation. However, these neurons are extremely fragile and survive for only a short time. OBJECTIVE： To establish a trypsin dissociation and culture method for studying neonatal rat cochlear spiral ganglion neurons. DESIGN： A single sample study. SETTING： Department of Otolaryngology, Head and Neck Surgery, Xijing Hospital, Fourth Military Medical University of Chinese PLA. MATERIALS： This study was performed at the central laboratory for Department of Otolaryngology, Head and Neck Surgery, Xijing Hospital, Fourth Military Medical University of Chinese PLA from February to May 2006. A total of 40 neonatal Sprague Dawley rats of either gender, aged 2-5 days, were provided by the Laboratory Animal Center of the Fourth Military Medical University of Chinese PLA. Trypsin and neuronal-specific nuclear protein （NeuN） monoclonal antibodies were purchased from Sigma Company, USA. Culture medium was synthesized using Dulbecco＇s modified Eagle＇s medium （DMEM）/F12 （Gibco Company, USA） supplemented with 10% fetal bovine serum （Sigma Company, USA）, 100 000 U/L penicillin, and 1 mol/L NaOH. The following protocol was performed in accordance with ethical guidelines for the use and care of animals. METHODS： After anesthesia, rats were sacrificed by neck dislocation. A complete cochlear axis with spiral ganglion tissue was removed. The cochlear axis was rinsed three times in a culture dish with a diameter of 35 mm using Hank＇s balanced solution. After washings, the tissue was cut into pieces, digested with 0.25% trypsin for about 20 minutes, and incubated in a 37 ℃ water bath. The tissue was centrifuged, then mixed with serum-containing culture medium. Using a transfer pipette, the cell suspension was transferred to polylysine （0.1%）-treated culture dishes with a diameter of 35 mm. The culture dish was incubated at 37 ℃, with a 5% CO2-air environment. Once the cells adhered to the culture dish wall, DMEM/F12 supplemented with 10% fetal bovine serum was added. MAIN OUTCOME MEASURE： Using an inverted microscope, the adherent cultured cells were observed and neurite growth index was calculated. Immunocytochemistry was performed to identify the spiral ganglion neurons, and NeuN-positive cells were analyzed. Following immunofluorescence, cochlear spiral ganglion neurons were identified through a microscope. RESULTS： Observation of cellular morphology： after digestion, inoculated cells exhibited were spherical, well stacked, and had a transparent appearance. Six hours later, some cells adhered to the culture dish wall, and small neurites were detected in a small number of cells. Twelve hours later, the adherent cells developed into polarized cells. Eighteen hours after inoculation, the adherent cells presented an ellipsoidal appearance, clear cell membranes, homogeneous cytoplasm, good refraction, and a transparent cell body surrounded by a marked halation. Twenty-four hours later, most of the cochlear spiral ganglion neurons exhibited a bipolar neuronal morphology with neurite length ranging from 2-5 times the length of a cell body. Some cochlear spiral ganglion neurons exhibited a tripolar neuronal morphology with neurites that stretched in three directions; neurite length was several times greater than the transverse diameter. Forty-eight to seventy-two hours later, the cells further differentiated and exhibited interwoven neurites, with a length that was 7-8 times greater than the cell body length. Seven days later, cells began to degenerate and underwent apoptosis. Identification of cochlear spiral ganglion neurons： immunocytochemical staining revealed whole cochlear spiral ganglion neurons that were green-colored and exhibited an ellipsoidal cell body with clear neurites. Measurement of neurite growth index： neurite growth index was 0.52±0.13, 0.86±0.21, 1.22±0.33, and 1.05 ±0.26 for 24, 48, 72, and 120 hours after inoculation, respectively. CONCLUSION： Under cell culture conditions of serum and trypsin dissociation, neonatal rat cochlear spiral ganglion neurons grow well, survive for long periods in vitro, and exhibit normal phenotypic differentiation.

Abnormal Innervation,Demyelination,and Degeneration of Spiral Ganglion Neurons as Well as Disruption of Heminodes are Involved in the Onset of Deafness in Cx26 Null Mice

GJB2 gene mutations are the most common causes of autosomal recessive non-syndromic hereditary deafness.For individuals suffering from severe to profound GJB2-related deafness,cochlear implants have emerged as the sole remedy for auditory improvement.Some previous studies have highlighted the crucial role of preserving cochlear neural components in achieving favorable outcomes after cochlear implantation.Thus,we generated a conditional knockout mouse model(Cx26-CKO)in which Cx26 was completely deleted in the cochlear supporting cells driven by the Sox2 promoter.The Cx26-CKO mice showed severe hearing loss and massive loss of hair cells and Deiter’s cells,which represented the extreme form of human deafness caused by GJB2 gene mutations.In addition,multiple pathological changes in the peripheral auditory nervous system were found,including abnormal innervation,demyelination,and degeneration of spiral ganglion neurons as well as disruption of heminodes in Cx26-CKO mice.These findings provide invaluable insights into the deafness mechanism and the treatment for severe deafness in Cx26-null mice.

Inhibition of the NLRP3 inflammasome attenuates spiral ganglion neuron degeneration in aminoglycoside-induced hearing loss

Aminoglycosides are a widely used class of antibacterials renowned for their effectiveness and broad antimicrobial spectrum.However,their use leads to irreversible hearing damage by causing apoptosis of hair cells as their direct target.In addition,the hearing damage caused by aminoglycosides involves damage of spiral ganglion neurons upon exposure.To investigate the mechanisms underlying spiral ganglion neuron degeneration induced by aminoglycosides,we used a C57BL/6J mouse model treated with kanamycin.We found that the mice exhibited auditory deficits following the acute loss of outer hair cells.Spiral ganglion neurons displayed hallmarks of pyroptosis and exhibited progressive degeneration over time.Transcriptomic profiling of these neurons showed significant upregulation of genes associated with inflammation and immune response,particularly those related to the NLRP3 inflammasome.Activation of the canonical pyroptotic pathway in spiral ganglion neurons was observed,accompanied by infiltration of macrophages and the release of proinflammatory cytokines.Pharmacological intervention targeting NLRP3 using Mcc950 and genetic intervention using NLRP3 knockout ameliorated spiral ganglion neuron degeneration in the injury model.These findings suggest that NLRP3 inflammasome-mediated pyroptosis plays a role in aminoglycoside-induced spiral ganglion neuron degeneration.Inhibition of this pathway may offer a potential therapeutic strategy for treating sensorineural hearing loss by reducing spiral ganglion neuron degeneration.

Recent advances in the application of MXenes for neural tissue engineering and regeneration

Transition metal carbides and nitrides(MXenes)are crystal nanomaterials with a number of surface functional groups such as fluorine,hydroxyl,and oxygen,which can be used as carriers for proteins and drugs.MXenes have excellent biocompatibility,electrical conductivity,surface hydrophilicity,mechanical properties and easy surface modification.However,at present,the stability of most MXenes needs to be improved,and more synthesis methods need to be explored.MXenes are good substrates for nerve cell regeneration and nerve reconstruction,which have broad application prospects in the repair of nervous system injury.Regarding the application of MXenes in neuroscience,mainly at the cellular level,the long-term in vivo biosafety and effects also need to be further explored.This review focuses on the progress of using MXenes in nerve regeneration over the last few years;discussing preparation of MXenes and their biocompatibility with different cells as well as the regulation by MXenes of nerve cell regeneration in two-dimensional and three-dimensional environments in vitro.MXenes have great potential in regulating the proliferation,differentiation,and maturation of nerve cells and in promoting regeneration and recovery after nerve injury.In addition,this review also presents the main challenges during optimization processes,such as the preparation of stable MXenes and long-term in vivo biosafety,and further discusses future directions in neural tissue engineering.

红景天苷改善顺铂引起的小鼠耳蜗毛细胞和螺旋神经节神经元损伤的机制

目的探究红景天苷(SAL)改善顺铂(CIS)引起的耳蜗毛细胞(CHC)和螺旋神经节神经元(SGN)损伤的作用及其与环磷腺苷(cAMP)/蛋白激酶A(PKA)/cAMP效应元件结合蛋白(CREB)通路的关系。方法分离新生C57BL/6小鼠的耳蜗基底膜,分为对照组(C组)、CIS组、SAL组、SAL+SQ22536(cAMP抑制剂)组和SAL+H-89(PKA抑制剂)组,每组20条。C组仅加入无血清BME培养液;CIS组在培养液中加入15μmol/L CIS;SAL组在CIS组基础上加入5μmol/L SAL;SAL+SQ22536组在CIS组基础上加入5μmol/L SAL和5μmol/L SQ22536;SAL+H-89组在CIS组基础上加入5μmol/L SAL和30μmol/L H-89。各组在培养箱中孵育48 h后,免疫荧光染色观察各组CHC和SGN损伤;试剂盒检测各组耳蜗基底膜中ROS和cAMP含量;Western blot检测各组PKA、p-CREB、CREB、Bcl-2、BDNF、NF-M蛋白水平。结果CIS组CHC排列混乱、体积肿大,SGN细胞核破碎、神经突缺失,SAL可减轻CHC和SGNs损伤。与C组相比,CIS组CHC、SGN数量较少(P<0.05),ROS、cAMP含量、PKA、BDNF、NF-M、Bcl-2蛋白及p-CREB/CREB水平较高(P<0.05);与CIS组相比,SAL组CHC、SGN数量较多(P<0.05),ROS含量较低(P<0.05),cAMP含量、PKA、BDNF、NF-M、Bcl-2蛋白及p-CREB/CREB水平较高(P<0.05)。SQ22536和H-89均可逆转SAL对CHC和SGN的保护作用。结论SAL可能通过激活cAMP/PKA/CREB通路,促进抗凋亡蛋白和神经保护因子表达,缓解CIS引起的CHC和SGN损伤。

基于自噬途径探讨银杏叶提取物对老年性聋大鼠的保护作用

目的探讨银杏叶提取物对老年性聋大鼠听功能、耳蜗组织形态学和自噬相关蛋白表达的作用。方法将45只大鼠随机分为对照组、模型组和银杏叶提取物低、中、高剂量组(10、20、30 mg/kg),每组9只,采用腹腔注射500 mg/kg D-半乳糖(D-gal)构建老年性聋模型。给药8周后,听性脑干诱发电位(ABR)检测大鼠听阈变化,HE染色观察耳蜗毛细胞、血管纹和螺旋神经节细胞形态变化,免疫荧光染色法检测耳蜗内外毛细胞数量变化,透射电子显微镜观察耳蜗毛细胞超微结构变化,Western blot法检测耳蜗组织中自噬相关蛋白表达。结果与对照组比较,模型组ABR阈值升高(P<0.01),耳蜗内外毛细胞、螺旋神经节细胞和血管纹损伤严重,血管纹厚度、螺旋神经节细胞数、内外毛细胞数和自噬小体数均降低(P<0.01),耳蜗组织Beclin1、LC3Ⅱ蛋白表达和LC3Ⅱ/LC3Ⅰ比值均降低(P<0.01),P62蛋白表达升高(P<0.01);与模型组比较,银杏叶提取物中、高剂量组ABR阈值均降低(P<0.01),耳蜗内外毛细胞及血管纹形态改善,螺旋神经节细胞形态趋向正常,血管纹厚度、螺旋神经节细胞数、内外毛细胞数和自噬小体数均增加(P<0.05,P<0.01),耳蜗组织Beclin1、LC3Ⅱ蛋白表达和LC3Ⅱ/LC3Ⅰ比值均升高(P<0.01),P62蛋白表达降低(P<0.01)。结论银杏叶提取物对老年性聋大鼠的听功能和耳蜗细胞具有保护作用,其机制可能与上调耳蜗组织Beclin1、LC3Ⅱ蛋白表达,下调P62蛋白表达有关。

PTC-209诱导体外螺旋神经元损伤作用及机制

目的 探讨Bmi1特异性抑制剂PTC-209诱导体外培养耳蜗器官螺旋神经元(SGNs)、听觉神经纤维(ANFs)的损伤作用,并分析其可能的调控机制。方法 选用P3新生SD仔鼠进行耳蜗器官培养,首先与不同浓度PTC-209进行培养,确定损伤模型(PTC-209组),即10μM PTC-209与耳蜗器官共培养48 h。随后将10μM PTC-209与5 mM GSH共培养,确定最佳保护模型(GSH+PTC-209组),即Pre6h 5 mM GSH对10μM PTC-209培养48 h。最后将实验分为Ctrl组、GSH组、PTC-209组及GSH+PTC-209组,培养结束后予MitoSOXRed进行组织染色,激光共聚焦显微镜观察。结果 PTC-209诱导耳蜗SGNs和ANFs损伤随浓度增高和作用时间延长而加重,其损伤作用呈时间和浓度依赖性。不同时间预处理GSH对PTC-209诱导SGNs和ANFs损伤存在差异保护作用,将各组的ANFs进行统计分析发现Pre 6 h GSH 5 mM+PTC-209 10μM组与其余各组ANFs差异均有统计学意义(P<0.01),提示Pre 6 h 5 mM GSH对10μM PTC-209培养48 h诱导SGNs和ANFs的损伤有显著保护作用。MitoSOX Red染色显示Ctrl组、GSH组及GSH+PTC-209组均未见红色荧光标记的ROS在SGNs、支持细胞(SCs)和ANFS蓄积;PTC-209组红色荧光标记的ROS在SGNs、SCs和ANFS大量蓄积。统计分析显示PTC-209组和Ctrl组、GSH+PTC-209组ROS的红色荧光光密度值差异均有统计学意义(q分别为343.5、349.3,P<0.01)。结论 PTC-209可诱导SGNs处细胞内的自由基和线粒体中ROS大量蓄积,进而导致氧化还原失衡,促进SGNs死亡,但是调控机制尚需要进一步探讨。

Basic flbroblast growth factor protects auditory neurons and hair cells from noise exposure and glutamate neurotoxicity

The purpose of the present study was to determine protectivie effects of basic fibroblast growth factor (bFGF) on cochlear neurons and hair cells in vitro and in vivo. In experiment I, cultured spiral ganglion neurons (SGNs) prepared from P3 mice were exposed to 20mM glutamate for 2 hours before the culture medium was replaced with fresh medium containing 0, 25, 50, and 100 ng/ml bFGF, respectively. Fourteen days later, all cultures were fixed with 4% paraformaldehyde, and stained with 1% toluidine blue. The number of surviving SGNs were counted and the length of SGNs neurites were measured. Exposure to 20 mM glutamate for 24 hours resulted in an inhibition on neurite outgrowth of SGNs and elevated cell death. Treatment of the cultures with bFGF led to promotion of neurite outgrowth and elevated number of surviving SGNs. Effects of bFGF were dose dependent with the highest potency at 100 ng/ml. In experiment Ⅱ, in vivo studies were carried out with guinea pigs in which bFGF or artificial perilymph was perfused into the cochlea to assess possible protective effects of bFGF on cochlear hair cells and compound action potentials(CAP). The CAPs were measured before, immediatly and 48 hours after exposure to noise. Significant differences in CAP were observed (p＜0. 05 ) among the bFGF perfused group, control group(t =3. 896 ) and artificial perilymph perfused group (t =2. 520) at 48 hours after noise exposure, Cochleae were removed and hair cell Loss was analyzed in surface preparations prepared from all experimental animals. Acoustic trauma caused loss of 651 and 687 inner hair cells in the control and artificial perilymph perfused group, respectively. In sharp contrast, only 31 inner hair cells were lost in the bFGF perfused ears. Similarly, more outer hair cells died in the control and perilymph perfuesed group (41830 and 41968, respectively) than in the group treated with bFGF (34258). Our results demonstrate that bFGF protected SGNs against glutmate neurotoxicity in vitro. In addition, treatment with bFGF also protected hair cells from acoustic trauma.

Inhibition of histone methyltransferase PRMT5 attenuates cisplatininduced hearing loss through the PI3K/Akt-mediated mitochondrial apoptotic pathway

This study aimed to evaluate the therapeutic potential of inhibiting protein arginine methyltransferase 5(PRMT5)in cisplatin-induced hearing loss.The effects of PRMT5 inhibition on cisplatin-induced auditory injury were determined using immunohistochemistry,apoptosis assays,and auditory brainstem response.The mechanism of PRMT5 inhibition on hair cell survival was assessed using RNA-seq and Cleavage Under Targets and Tagment-quantitative polymerase chain reaction(CUT&Tag-qPCR)analyses in the HEI-OC1 cell line.Pharmacological inhibition of PRMT5 significantly alleviated cisplatin-induced damage to hair cells and spiral ganglion neurons in the cochlea and decreased apoptosis by protecting mitochondrial function and preventing the accumulation of reactive oxygen species.CUT&Tag-qPCR analysis demonstrated that inhibition of PRMT5 in HEI-OC1 cells reduced the accumulation of H4R3me2s/H3R8me2s marks at the promoter region of the Pik3ca gene,thus activating the expression of Pik3ca.These findings suggest that PRMT5 inhibitors have strong potential as agents against cisplatininduced ototoxicity and can lay the foundation for further research on treatment strategies of hearing loss.

NT-3对耳蜗螺旋神经节发育的调控机制研究进展

耳蜗螺旋神经元(spiral ganglion neurons,SGN)的存活和增殖依赖于各种神经营养因子的调节。SGN的损伤和发育异常均可导致感音神经性耳聋,但SGN无再生能力,听力下降往往很难恢复。因此,对听力下降进行早期干预,防止其不可逆的损伤尤为重要。神经营养素3(neurotrophin-3,NT-3)在耳蜗中高表达并促进SGN的存活、轴突的生长和损伤的修复,这已得到广泛的共识,但对其如何将信息传递到细胞内,从而调节SGN的发育仍然不清楚。本文就NT-3在SGN发育期中作用机制的研究进展作一综述。

Waardenburg综合征患者螺旋神经节细胞数量分布特征的分析

目的本研究旨在研究Waardenburg综合征(Waardenburg Syndrome,WS)患者的耳蜗中螺旋神经节细胞(spiral ganglion neuron,SGN)的数量与非综合征性感音神经性聋(Nonsyndromic sensorineural hearing loss,NS)患者相比是否具有差异,以及耳蜗不同位置之间的数量分布差异。方法本研究采用横断面研究,收集2000年至2020年间就诊于中国人民解放军总医院行人工耳蜗植入的WS患者,收集患者相关信息,包括植入年龄、植入侧别、植入电极型号以及人工耳蜗植入术中的电刺激复合动作电位(Electrical compound action potential,ECAP)测量数据以及其幅值增长曲线(amplitude growth function,AGF)。为控制植入年龄,植入电极类型等混杂因素带来的偏倚,制定对照组患者纳入标准,通过采用窗函数的计算方式,应用Matlab(R2016a)计算两组中不同刺激电极的slope值。依据刺激电极在耳蜗中的位置,将电极分为近蜗顶组,中间组以及近蜗底组,应用采用t检验比较两组患者中不同位置之间的slope值。结果依据纳入以及排除标准,最终纳入16名WS患者以及16名NS患者,经两独立样本t检验证实两组之间具有可比性。WS患者与NS患者的总体slope具有显著差异(P=0.007<0.05;Cohen's d=1.022>1),在不同位置中,以中间组以及近蜗底组的slope值具有显著差异(P=0.001<0.05;P=0.026<0.05,Co⁃hen's d=1.302>1;Cohen's d=0.828>0.8)。结论WS患者SGN的数量明显少于NS患者,并且在耳蜗的不同位置数量改变不同,尤其以底回以及中回的数量改变尤为显著。

α2-肾上腺素能受体在水杨酸钠诱导大鼠耳蜗螺旋神经节神经元兴奋毒性中的表达变化

目的 观察α2-肾上腺素能受体(α2-adrenergic receptor, α2-AR)在大鼠耳蜗螺旋神经节神经元(spiral ganglion neuron, SGN)中的分布,以及水杨酸钠(sodium salicylate, SS)作用SGN不同时间点后α2-AR各亚型的表达改变,以探讨其在SGN兴奋毒性中的作用。方法 将新生SD大鼠随机分成:对照组、SS处理6 h组、SS处理12 h组和SS处理24 h组,每组2只。急性分离各组SGN后进行原代培养48 h,对照组用新的培养基培养,不做处理,余三组分别用5 mM水杨酸钠处理6 h、12 h、24 h,用免疫荧光染色技术检测SGN中α2-AR各亚型的表达情况;采用实时荧光定量PCR(RT-qPCR)和Western blot方法检测SS作用不同时间后α2-AR各亚型的mRNA和总蛋白表达改变情况。结果 (1)免疫荧光染色表明SGN上存在α2A-AR、α2B-AR、α2C-AR三种亚型的表达;(2)RT-qPCR结果显示在SGN中α2-AR各亚型mRNA相对表达量α2A-AR>α2B-AR(P<0.01),α2A-AR>α2C-AR(P<0.01),α2B-AR和α2C-AR之间差异无统计学意义;与对照组相比,5 mM SS作用SGN 6 h、12 h、24 h后α2A-AR表达量均明显增高(P<0.05);SS作用12 h后α2C-AR表达量开始增高(P<0.05),而α2B-AR呈升高趋势,但差异无统计学意义(P>0.05);(3)Western blot结果显示SGN中α2-AR各亚型蛋白表达量α2A-AR>α2B-AR(P<0.01),α2A-AR>α2C-AR(P<0.05),α2B-AR和α2C-AR之间差异无统计学意义,与mRNA表达一致。与对照组相比,5 mM SS作用SGN 12 h后α2A-AR总蛋白开始下降(P<0.05),作用24 h后α2C-AR总蛋白下降(P<0.05),α2B-AR总蛋白不变。结论 α2-AR三种亚型在SGN中均有表达,其中α2A-AR、α2C-AR可能介导了SS诱导的SGN兴奋毒性且α2A-AR比α2C-AR反应更快速。

艾塞那肽减轻卡那霉素加呋塞米诱导的小鼠螺旋神经元损伤

目的探讨高血糖素样肽-1受体(glucagon like peptide-1 receptor,GLP-1R)激动剂艾塞那肽(exendin-4)对卡那霉素加呋塞米诱导的小鼠螺旋神经元(spiral ganglion neurons,SGNs)损伤的作用。方法原代培养的小鼠SGNs进行免疫荧光染色、小鼠耳蜗切片免疫组化染色,验证GLP-1R在SGNs上的表达。将30只C57BL/6J小鼠分成3组,每组10只,卡那霉素加呋塞米组一次性腹腔注射硫酸卡那霉素1 g/kg及呋塞米0.4 g/kg;艾塞那肽组卡那霉素和呋塞米注射前3 d开始腹腔注射艾塞那肽400μg/kg,每天1次,直到卡那霉素和呋塞米给药后第7 d;对照组腹腔注射等量生理盐水。分组处理后,使用HE染色观察SGN损伤情况并计数骨螺旋板里SGNs数量;采用Western blot技术检测各组耳蜗组织GLP-1R及凋亡相关蛋白Bax、Bcl-2、Cleaved-caspase-3的表达。结果免疫荧光及免疫组化染色结果显示,GLP-1R表达于SGNs。HE染色显示,与对照组比较,卡那霉素加呋塞米组SGNs数量下降,艾塞那肽组SGNs数量有所恢复(P<0.05);Western blot结果显示,卡那霉素加呋塞米组GLP-1R蛋白表达下降,促凋亡蛋白Cleaved-caspase-3、Bax蛋白水平升高,抗凋亡蛋白Bcl-2表达水平下降;艾塞那肽组GLP-1R蛋白表达较卡那霉素加呋塞米组升高,Cleaved-caspase-3、Bax表达降低,Bcl-2表达升高(P<0.05)。结论GLP-1R激动剂艾塞那肽可能通过减轻细胞凋亡,拮抗卡那霉素加呋塞米诱导的小鼠SGNs损伤。

ApoE敲除导致小鼠耳蜗螺旋神经元功能损伤

目的:探究载脂蛋白E(apolipoprotein E,ApoE)基因缺陷引起小鼠耳蜗螺旋神经节处听神经损伤的可能机制。方法:对ApoE基因敲除(ApoE-/-)小鼠进行听性脑干反应(auditory brainstem response,ABR)检测,并分析波群振幅及延迟期。免疫荧光及HE染色分析耳蜗螺旋神经节内听神经及带状突触的病理损伤情况。蛋白质印迹、q RT-PCR等实验检测Lim激酶1(LIM domain kinase 1,Limk1)、丝切蛋白1(cofilin1)蛋白表达及磷酸化水平,并检测磷脂酰肌醇3激酶(phosphatidylinositide 3-kinases,PI3K)通路激活情况。结果:ApoE-/-小鼠ABR检测中波Ⅰ的振幅缩短,延迟期延长。螺旋神经节处染色结果显示ApoE-/-小鼠带状突触及听神经纤维数量减少,并伴随结构受损。Western blot、qRT-PCR及免疫荧光染色发现Limk1与cofilin1在ApoE敲除后蛋白磷酸化水平出现显著性下降,同时PI3K通路激活被抑制。结论:ApoE基因缺陷会引起耳蜗螺旋神经节处带状突触及听神经损伤,并可能与神经元肌动蛋白细胞骨架密切相关。

老年性耳聋病因及分子机制的研究进展

老年性耳聋是老年人群中最常见的感觉缺陷之一,又称为年龄相关性听力损失,是老年人最普遍的感觉缺陷。随着社会逐渐老龄化,老年性耳聋的发病率越来越高。老年性耳聋严重影响了老年人的身心健康,导致老年人的生活质量下降、认知能力改变、社交孤立以及抑郁、沟通障碍等。研究其病因及发病机制对老年性耳聋的诊断、治疗和预防具有重要意义。本综述总结了老年性耳聋分子机制、致病因素、病理分型、治疗手段等。

水杨酸钠增强氧化应激介导耳蜗螺旋神经节神经元损伤作用的实验观察

目的观察水杨酸钠(SS)作用大鼠耳蜗螺旋神经节神经元(SGN)后,SGN中氧化应激水平的变化。方法将6只SD大鼠随机分为对照组和SS组,每组3只。通过耳蜗器官培养48 h后,用5 mM SS处理48 h,对照组不予处理,采用免疫荧光染色技术定位耳蜗器官中活性氧自由基(ROS)的主要生成位置。将15只SD大鼠随机分成5组,即对照组、SS组、SS+N-乙酰-L-半胱氨酸(NAC)组、阳性对照过氧化氢(H_(2)O_(2))组、H_(2)O_(2)+NAC组,每组3只。通过急性分离SGN,原代培养48 h后分别用5 mM SS、5 mM SS联合100μM NAC、300μM H_(2)O_(2)、300μM H_(2)O_(2)联合100μM NAC处理48 h,对照组不予处理。采用荧光染色法检测并量化各组大鼠SGN中ROS荧光探针DCFH-DA的平均荧光强度;采用CCK8法检测SGN细胞存活率。结果在耳蜗器官培养中,SS作用后,免疫荧光染色显示ROS荧光增强,并且主要在SGN中表达,而在其他细胞中荧光强度无明显变化。为进一步量化荧光强度,在原代培养SGN中加入5 mM SS处理后,荧光染色法显示ROS平均荧光强度较对照组升高(P<0.001),与H 2O 2组结果一致(P<0.0001)。在加入ROS抑制剂NAC之后,ROS平均荧光强度较SS组下降(P<0.01)。CCK8法结果显示,SS作用之后细胞存活率较对照组下降41.34%(P<0.01);在加入NAC之后,细胞存活率较SS组上升36.05%(P<0.01),与对照组比较差异无统计学意义(P>0.05)。H_(2)O_(2)组细胞存活率较对照组下降52.31%(P<0.001),在加入NAC之后,细胞存活率较H_(2)O_(2)组上升34.73%(P<0.01)。结论SS增强SGN的氧化应激,并且导致了SGN损伤。氧化应激抑制剂NAC可以降低SGN的氧化应激,并且对SS致SGN损伤具有保护作用。